Zinsi polypeptide composition stimulating pancreatic islet growth

ABSTRACT

The present invention provides compositions for stimulating an increase in islet proliferation and β-cell mass using an insulin homolog polypeptide. The present invention also includes methods for treating diabetes by stimulating islet proliferation and β-cell mass increases and affecting insulin levels.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.08/991,890, filed Dec. 16, 1997, now U.S. Pat. No. 6,114,307, issued onSep. 5, 2000, which is related to U.S. Provisional Patent ApplicationSerial No. 60/033,003, filed on Dec. 16, 1996. Under 35 U.S.C §§119(e)(1) and 120, this application claims benefit of said applicationsand patent.

BACKGROUND OF THE INVENTION

β-cells are specialized cells that secrete insulin and are found inpancreatic islets. Insulin belongs to a group of protein/polypeptidehormones. Insulin increases the rate of synthesis of glycogen, fattyacids, and proteins and stimulates glycolysis and cell proliferation. Italso promotes the transport of glucose, and some other sugars, and aminoacids into muscle and fat cells. Insulin levels are regulated tomaintain glycemic homeostasis, and an important mechanism for regulatinginsulin production, and hence insulin levels, is β-cell mass.

During the lifetime of an individual metabolic needs can changedrastically, requiring dynamic changes in cells and tissues thatregulate homeostasis. During pregnancy (Marynissen et al., Diabetes36:883-891, 1987) β-cell mass increases, as well as in response toobesity (Kloppel et al., Surv. Synth. Pathol. Res. 4:110-125, 1985).These increases in β-cell mass are attributed to an increasedrequirement for insulin to maintain normal glucose levels (Parsons etal., Endocrinology 130:1459-1466, 1992). It has also been shown thatβ-cell mass normally decreases post-partum, primarily by apoptosis(Scaglia et al., Endocrinology 136:5461-5468, 1995).

It is generally believed that increases in β-cell mass occurs in threeways: 1) an increase in cell size and function; 2) increasedproliferation of mature β-cells; and/or 3) increased,recruitment anddifferentiation of β-cell progenitors. In diabetic mice, animals thatreceived islet transplants and then achieved normal glycemia, showedβ-cell hypertrophy, rather than an increase in cell replication (Montanaet al., J. Clin. Invest. 91:780-787, 1993). Adult β-cell regenerationhas been demonstrated in rodents (Hellerstrom et al., in “The Pathologyof the Endocrine Pancreas in Diabetes”, P. J. Lefebvre and D. G.Pipeleers, eds., pp. 141-170, Springer-Verlag, Heidelberg, 1988). Inpartially pancreatectomized rats both preexisting β-cells, as well asproliferation and differentiation of precursor cells, have beendemonstrated to expand (Bonner-Weir, Diabetes Nutr. Res. 5, Supp.1:21-25, 1992).

Several factors have been shown to increase β-cell mass. These factorsinclude glucose (Woerner, Anal. Rev. 71:33-57, 1938), IGF-I (Rabinovitchet al., Diabetes 31:160-164, 1982), reg protein (Terazono et al., J.Biol. Chem. 263:2111, 1988) and possibly a combination of TGF-α andgastrin (Bonner-Weir, Recent Prog. Hormone Res. 12:91-104, 1994). Whilesome factors have been shown to increase β-cell mass in vitro or invivo, understanding of the process is poorly understood and thepossibility that other unidentified factors are involved is likely.

Recently a new member of the insulin superfamily has been identified,early placenta insulin-like factor or placentin (Chassin et al.,Genomics 29:465-470, 1995). Placentin cDNA was isolated from firsttrimester human placenta and found to have a 139-amino acid open readingframe. Based on homology to the rest of the insulin superfamily it waspredicted that placentin, like preprorelaxin and preproinsulin, wouldhave a signal sequence, followed by the B chain, C peptide, A chain. Themature molecule would have the signal peptide and C peptide removed,with the B and A chains joined by both inter- and intra-chain disulfidebonds (Chassin et al., 1995, ibid. and James et al., Nature 267:544-546,1977). The B-chain, C-peptide, A-chain motif is found in several otherproteins, including relaxin (U.S. Pat. No. 4,835,251), insulin-likegrowth factors (IGF) I and II (Bang and Hall, in “Insulin-like GrowthFactors”, P. N. Schofield (ed.), pp. 151-177, Oxford University Press,Oxford, 1992), and Leydig Factor (Bullesbach et al., J. Biol. Chem.270:16011-16015, 1995). Unlike other members of the insulin superfamily,IGF I and IGF II have D and E domains that are cleavedpost-translationally. Cysteines that are involved in disulfide bonds areconserved in.all the members of the family and play a role in thetertiary structure of the molecules.

Placentin has been shown to stimulate ³H-thymidine uptake in humanplacental 3AsubE cells and stimulate human chorionic gonadotropinproduction in primary cultures of trophoblasts (Koman et al., J. Biol.Chem. 271:20238-20241, 1996). This activity suggests that placentin mayplay a role during placental development. However, the presentinventors, surprisingly, have found that a molecule encoded by the DNAfor placentin, but a different amino acid structure, increases β-cellmass and may be useful in treatment of diabetes, and further that thebiologically active molecule differs from the molecule described in theart.

SUMMARY OF THE INVENTION

The present invention provides proteins produced by a method comprising:culturing a host cells into which has been introduced a DNA expressionvector comprising a transcription promoter; a DNA segment comprising anucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 76 tonucleotide 417; and a transcription terminator, wherein said host cellexpresses the polypeptide encoded by said DNA segment and recoveringsaid protein.

In another embodiment, the host is a mammalian cell. In anotherembodiment, the host has had a second DNA expression vector introducedinto it, wherein the second expression vector comprises a transcriptionpromoter; a DNA segment encoding an endoprotease; and a transcriptionterminator, wherein said host cell expresses the a DNA segmentcomprising a nucleotide sequence as shown in SEQ ID NO: 1 fromnucleotide 76 to nucleotide 417 and said DNA segment encoded by theendoprotease.

In another aspect, the present invention provides an isolated andpurified protein comprising a first polypeptide comprising an amino acidsequence as shown in SEQ ID NO: 2 from residue 26 (Ala) to residue 110(Ser) or 114 (Arg); and a second polypeptide comprising an amino acidsequence as shown in SEQ ID NO: 2 from residue 115 (Ser) to residue 139(Thr), wherein said first polypeptide and said second polypeptide arecapable of disulfide associating.

In another aspect, the present invention provides an isolated andpurified protein comprising a first polypeptide comprising an amino acidsequence as shown in SEQ ID NO: 2 from residue 26 (Ala) to residue 48(Lys), 49 (Thr) or 50 (Phe); and a second polypeptide comprising anamino acid sequence as shown in SEQ ID NO: 2 from residue 115 (Ser) toresidue 139 (Thr), wherein said first polypeptide and said secondpolypeptide are capable of disulfide associating.

In another aspect, the present invention provides a method ofstimulating proliferation of pancreatic islet comprising administeringto a mammal in need thereof, an amount of an isolated and purifiedpolypeptide comprising: a first polypeptide comprising an amino acidsequence as shown in SEQ ID NO: 2 from residue 26 (Ala) to residue 110(Ser) or 114 (Arg); and a second polypeptide comprising an amino acidsequence as shown in SEQ ID NO: 2 from amino acid residue 115 (Ser) toresidue 139 (Thr), and wherein said first polypeptide and said secondpolypeptide are capable of disulfide associating, sufficient to producea clinically significant increase in insulin secretory capacity.

In another aspect, the present invention provides a method ofstimulating proliferation of pancreatic islets comprising administeringto a mammal in need thereof, an amount of an isolated and purifiedpolypeptide comprising: a first polypeptide comprising an amino acidsequence as shown in SEQ ID NO: 2 from residue 26 (Ala) to residue 48(Lys), 49 (Thr) or 50 (Phe); and a second polypeptide comprising anamino acid sequence as shown in SEQ ID NO: 2 from amino acid residue 115(Ser) to residue 139 (Thr), and wherein said first polypeptide and saidsecond polypeptide are capable of disulfide associating, sufficient toproduce a clinically significant increase in insulin secretory capacity.

In other embodiments, the present invention provide methods wherein theclinically significant increase in insulin secretory capacity results ina decrease in fasting plasma glucose levels.

In other embodiments, the present invention provide methods wherein theisolated and purified protein is administered in combination with aninsulin sensitizer.

In another aspect, the present invention provides a method forstimulating in vitro proliferation of pancreatic islet cells comprisingculturing islets with an amount of an isolated and purified proteincomprising: a first polypeptide comprising an amino acid sequence asshown in SEQ ID NO: 2 from residue 26 (Ala) to residue 110 (Ser) or 114(Arg); and a second polypeptide comprising an amino acid sequence asshown in SEQ ID NO: 2 from amino acid residue 115 (Ser) to residue 139(Thr), and wherein said first polypeptide and said second polypeptideare capable of disulfide associating, sufficient to produce an increasein the number of islet cells as compared to islet cells cultured in theabsence of the protein.

In another aspect, the present invention provides a method forstimulating in vitro proliferation of pancreatic islet cells comprisingculturing islets with an amount of an. isolated and purified proteincomprising: a first polypeptide comprising an amino acid sequence asshown in SEQ ID NO: 2 from residue 26 (Ala) to residue 26 (Ala) toresidue 48 (Lys), 49 (Thr) or 50 (Phe); and a second polypeptidecomprising an amino acid sequence as shown in SEQ ID NO: 2 from aminoacid residue 115 (Ser) to residue 139 (Thr), and wherein said firstpolypeptide and said second polypeptide are capable of disulfideassociating, sufficient to produce an increase in the snumber of isletcells as compared to islet cells cultured in the absence of the protein.

In other embodiments, the present invention provides methods whereinsaid cells are cultured in 0.1 ng/ml to 100 ng/ml of said protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that animals treated with BHK cells transfected withzins1 have a 50% increase in islet numbers over animals that have beentreated with, untransfected BHK cells.

FIG. 2 illustrates that animals treated with BHK cells transfected with,zins1 have a trend toward increased islet size over animals treated withuntransfected BHK cells.

DETAILED DESCRIPTION OF THE INVENTION

Prior to describing the present invention in detail, it may be helpfulto define certain terms used herein:

The term “affinity tag” is used herein to denote a peptide segment thatcan be attached to a polypeptide to provide for purification of thepolypeptide or provide sites for attachment of the polypeptide to asubstrate. In principal, any peptide or protein for which an antibody orother specific binding agent is available can be used as an affinitytag. Affinity tags include a poly-histidine tract, protein A (Nilsson etal., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3,1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-1210,1988; available from Eastman Kodak Co., New Haven, Conn.), streptavidinbinding peptide, or other antigenic epitope or binding domain. See, ingeneral Ford et al., Protein Expression and Purification 2: 95-107,1991, which is incorporated herein by reference. DNAs encoding affinitytags are available from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

The term “allelic variant” denotes any of two or more alternative formsof a gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequence. The term allelic variant is also used herein to denote aprotein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides and proteins. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide or protein to denote proximity or relativeposition. For example, a certain sequence positioned carboxyl-terminalto a reference sequence within a protein is located proximal to thecarboxyl terminus of the reference sequence, but is not necessarily atthe carboxyl terminus of the complete protein.

The term “complements of polynucleotide molecules” denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a polynucleotide segment equivalent innucleotide sequence to an EST. A “contig assembly” denotes a collectionof EST contigs that define a larger polynucleotide segment containing anopen reading frame encoding a full-length or partial polypeptide.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide)Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” denotes a DNA molecule, linear or circular,that comprises a segment encoding a polypeptide of interest operablylinked to additional segments that provide for its transcription. Suchadditional segments may include promoter and terminator sequences, andmay optionally include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, and the like.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide molecule, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include CDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985).

When applied to a protein, the term “isolated” indicates that theprotein is found in a condition other than its native environment, suchas apart from blood and animal tissue. In a preferred form, the isolatedprotein is substantially free of other proteins, particularly otherproteins of animal origin. It is preferred to provide the protein in apurified form, i.e., greater than 95% pure, more preferably greater than99% pure.

The term “operably linked”, when referring to DNA segments, denotes thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in the promoter andproceeds through the coding segment to the terminator

The term “ortholog” (or “species homolog”) denotes a polypeptide orprotein obtained from one species that is the functional counterpart ofa polypeptide or protein from a different species. Sequence differencesamong orthologs are the result of speciation.

The term “paralog” denotes a polypeptide or protein obtained from agiven species that has homology to a distinct polypeptide or proteinfrom that same species.

The term “polynucleotide” denotes a single- or double-stranded polymerof deoxyribonucleotide or ribonucleotide bases read from the 5′ to the3′ end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared, from a combinationof natural and synthetic molecules.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Small polypeptidesare commonly referred to as “peptides”.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “promoter” denotes a portion of a gene containing DNA sequencesthat provide for the binding of RNA polymerase and initiation oftranscription. Promoter sequences are commonly,. but not always, foundin the 5′ non-coding regions of genes.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. Most nuclear receptors also exhibit amulti-domain structure, including an amino-terminal, transactivatingdomain, a DNA binding domain and a ligand binding domain. In general,receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

All references cited herein are incorporated by reference in theirentirety.

One aspect of the present invention is based in-part on the discoverythat an insulin-homolog DNA previously described as placentin encodes adifferent polypeptide from that described as the placentin protein.

Another aspect of the present invention provides methods foradministering the novel protein to stimulate pancreatic islet cells toproliferate in vivo and in vitro. Islet cell proliferation is a measureof increase in β-cell mass. Thus, the molecules of the present inventionprovide a means for increasing the size and number of β-cells (β-cellmass), and thereby increasing insulin availability.

The DNA sequence for placentin was reported to have a 139 amino acidcodon open reading frame (WO 95/34653 and Chassin et al., 1995, ibid.),and was predicted to encode a secretory signal sequence and a maturepolypeptide. The mature polypeptide was shown to have homology withinsulin, relaxin 1 and 2, and Leydig Factor, and thus, was considered amember of the insulin superfamily. Within this family, the cysteinemotif is highly conserved in the B and A chains, where the B chain motifcan be represented as LCGX{10}C, where X{ } is the number of any aminoacid residues except cysteine (as shown in SEQ ID NO: 6). The A chainmotif is CCX{3}CX{8}C, where X{ } is the number of any amino acidresidues, except cysteine (as shown in SEQ ID NO: 7).

Insulin is synthesized by β-cells of pancreatic islets as preproinsulin,and processing of the mature protein molecule involves cleavage at theC-terminus of the secretory signal polypeptide, and cleavage at theC-terminus of the B chain and at the N-terminus of the A chain,resulting in removal of the C-peptide. The cleavage sites for removal ofthe secretory signal peptide and C-peptide are not conserved within theinsulin superfamily. Chassin et al. (Genomics 29:465-470, 1995)disclosed that the predicted mature placentin molecule would be cleavedat a serine (amino acid residue 17 of SEQ ID NO: 2) to remove the signalpeptide and at leucine (amino acid residue 58 of SEQ ID NO: 2) andleucine (amino acid residue 109 SEQ ID NO: 2) to remove the C-peptide.

However, the present inventors predicted a different mature protein,which has been designated Zins1, comprising a disulfide-bonded B chainand A chain, wherein the B chain comprises the amino acid sequence ofSEQ ID NO: 2 from amino acid residue 26 (Ala) to at least amino acidresidue 43 (Cys) and wherein the A chain comprises the amino sequence ofSEQ ID NO: 2 from amino acid residue 115 (Ser) to residue 139 (Thr),based on sequence alignment and analyses. Furthermore, the presentinventors have isolated and purified the polypeptide from mediumconditioned by host cells co-expressing a first DNA construct comprisingthe sequence of, SEQ ID NO: 1 from nucleotide 1 to nucleotide 420 with asecond DNA construct encoding for endoprotease PC3. PC3 is one ofseveral endoproteases shown to be restricted to endocrine andneuroendocrine tissues and is involved in prohormone processing. TheZins1 protein has demonstrated biological activity that resulted inincreased β-cell mass and lowered blood glucose levels.

Analyses of the polypeptides present in the conditioned medium revealedpolypeptides comprising a first polypeptide comprising amino acidresidue 26 (Ala) to residue 110 (Ser) or 114 (Arg) as shown in SEQ IDNO: 2, and a second polypeptide comprising amino acid residue 115 (Ser)to residue 139 (Thr) as shown in SEQ ID NO: 2. These data suggest thatthe first polypeptide comprises a B chain and C-peptide and the secondpolypeptide comprises an A chain, wherein the first and secondpolypeptides are capable of disulfide associating.

Processing of the mature protein molecule involves cleavage at theC-terminus of the secretory signal peptide, and, based on predictedstructural homology with other mature members of the insulinsuperfamily, a cleavage at the C-terminus of the B chain and at theN-terminus of the A chain, resulting in removal of the C-peptide.Alignment of the deduced amino acid sequence of the Zins1 polypeptide ofthe present invention with other known members of the insulinsuperfamily predicts a signal peptide cleavage site at amino acidresidue 25 (Ala) of SEQ ID NO:2. Cleavage at the N-terminus of the Achain is predicted to be after amino acid residue 114 (Arg). Thecleavage site at the junction of the C-peptide and A chain is highlyconserved, occurring after Arg-X-X-Arg (wherein X is any amino acidresidue), Arg-Arg or Lys-Arg; however, the cleavage sites at thejunction of the signal sequence and B chain, and at the junction of theB chain and C-peptide, do not maintain a similarly high degree ofconservation within the insulin family.

The C-peptide portion of insulin superfamily members is highlydivergent. Also, the gene encoding Zins1 includes a silent intron ofabout 2 kb that interrupts the coding sequence in the C-peptide domain,adding another potential variable to the C-peptide portion of Zins1.Zins1 has a 139 amino acid open reading frame, compared to a 110 aminoacid open reading frame for human insulin and about 100-200 amino acidopen reading frames for other insulin superfamily members. Except forthe IGFs (which contain D and E domains), the open reading frame lengthvariations among the members of the insulin superfamily arepredominantly associated with C-peptide variations in length.

The enzymology of proinsulin conversion suggests that prohormoneconvertase 3 (PC3) cleaves primarily at the B chain-C-peptide junction,and that PC2 cleaves preferentially at the C-peptide-A-chain junctionand favors proinsulin already processed by PC3 over intact prohormone.In human and rat proinsulin, dibasic residues link the B chain andC-peptide and the C-peptide and A chain. In addition, a basic residue 4residues N-terminal to the cleavage site (a “P4 basic residue”) may bepresent at one or both junctions, and may influence the ability of PC3,PC2 or furin to cleave at the junction sites. In a study reported by F.Vollenweider et al. (Diabetes 44:1075-80, 1995), cotransfection of COScells with PC3 and either human proinsulin, rat proinsulin II or mutanthuman proinsulin Arg⁶² showed that PC3 cleaved both proinsulinjunctions, regardless of the presence or absence of a P4 basic residue.

Zins1 does not have basic or dibasic residues from position 49 (Thr) to62 (Gly), as shown in SEQ ID NO: 2. There is an Arg residue at position63, and a Lys residue at positions 65, 74, 94, 95, 105 and 106. AnArg-Lys-Lys-Arg motif is also present at residues 111-114, just beforethe A chain start sequence described by the resent invention. Based onsequence alignments, nowledge of prohormone conversion enzymes, and thedata resented herein, a B chain-C-peptide junction cannot bedefinitively determined. Chassin et al. (ibid.) describe a putativecleavage site at the junction of the B chain and C-peptide of placentin(INSL4) between residue 58 (Leu) and residue 59 (Leu) of SEQ ID NO:2.Koman et al. (ibid.) describe a putative cleavage site at the junctionof the B chain and C-peptide of placentin between residue 62 (Gly) andresidue 63 (Arg) of SEQ ID NO:2. However, neither group providesrationale or data to support cleavage at these sites.

Upon expression of the Zins1 molecule in mammalian cells, the presentinventors have discovered that the C-peptide is glycosylated.Carbohydrate analysis revealed that 1 O-glycosylation site is present ateither residue 49 (Thr), 50 (Thr), 51 (Thr) or 61 (Ser) of SEQ ID NO: 2.Based on homology with other members of the insulin superfamily, whereO-glycosylation has not been identified in any B-chain, the Bchain/C-peptide cleavage is predicted to occur after residue 48 (Lys) orresidue 49 (Thr) or residue 50 (Phe) of SEQ ID NO: 2, with theO-glycosylation occurring at one of residues 51, 52, 53 or 61. Two moreO-glycosylation sites are predicted at residues 69 (Ser), 70 (Thr),and/or 71 (Ser) of SEQ ID NO: 2. Another glycosylation site is predictedat either residue 81 (Thr), 82 (Thr), 83 (Ser) or 90 (Ser) of SEQ ID NO:2.

While not wanting to be bound by theory, the B chain/C-peptide form ofthe Zins1 molecule may have an important role in the biological functionof the molecule. The B chain/C-peptide may form a domain that isinvolved in directing the molecule to its target; processing of themolecule to its biologically active form; regulation of receptormultimerization and involvment in formation of tertiary structure, suchas folding. In addition, the C-peptide, particularly in light of theputative glycosylation sites, may function as an independent molecule.

Therefore, the present invention provides isolated Zins1 proteins thatare substantially homologous to the polypepitides of SEQ ID NO: 2. Inone embodiment, the isolated and purified Zins1 proteins comprise afirst polypeptide (B chain) comprising the amino acid sequence of SEQ IDNO: 2 from amino acid residue 26 (Ala) to at least amino acid residue 43(Cys) and a second polypeptide (A chain) comprising the amino sequenceof SEQ ID NO: 2 from amino acid residue 115 (Ser) to residue 139 (Thr),wherein said first and second polypeptides are capable of disulfideassociating; and their allelic variants and orthologs.

In another embodiment, the present invention provides isolated andpurified Zins1 proteins that comprise a first polypeptide comprising theamino acid sequence of SEQ ID NO: 2 from amino acid residue 26 (Ala) toan amino acid residue selected from the group consisting of 48 (Lys), 49(Thr) and 50 (Phe); and a second polypeptide comprising the aminosequence of SEQ ID NO: 2 from amino acid residue 115 (Ser) to residue139 (Thr), wherein said first and second polypeptides are capable ofdisulfide associating; and their allelic variants and orthologs.

In another embodiment, the present invention provides isolated andpurified Zins1 protein that comprise a first polypeptide comprising theamino acid sequence of SEQ ID NO: 2 from amino acid residue 26 (Ala) toamino acid residue 110 (Ser) or 114 (Arg) and a second polypeptidecomprising the amino sequence of SEQ ID NO: 2 from amino acid residue115 (Ser) to residue 139 (Thr), wherein said first and secondpolypeptides are capable of disulfide associating; and their allelicvariants and orthologs. Cleavage at the C-peptide/A chain junctionoccurs at residue 114 (Arg), but carboxypeptidases are well known toremove dibasic residues resulting in the final C-peptide C-terminusbeing between residue 114 (Arg) and residue 110 (Ser).

The term “substantially homologous” is used herein to denotepolypeptides having 50%, preferably at least 80%, more preferably 90%identical and most preferably 95% or more identity to the polypeptidesas shown in SEQ ID NO: 2.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 1 (amino acids are indicated by the standard one-lettercodes).

TABLE 1 A R N D C Q E G H I L K M F P S T W Y V A   4 R −1   5 N −2   0  6 D −2 −2   1   6 C   0 −3 −3 −3   9 Q −1   1   0   0 −3   5 E −1   0  0   2 −4   2   5 G   0 −2   0 −1 −3 −2 −2   6 H −2   0   1 −1 −3   0  0 −2   8 I −1 −3 −3 −3 −1 −3 −3 −4 −3   4 L −1 −2 −3 −4 −1 −2 −3 −4 −3  2   4 K −1   2   0 −1 −3   1   1 −2 −1 −3 −2   5 M −1 −1 −2 −3 −1   0−2 −3 −2   1   2 −1   5 F −2 −3 −3 −3 −2 −3 −3 −3 −1   0   0 −3   0   6P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4   7 S   1 −1   1   0 −1   0  0   0 −1 −2 −2   0 −1 −2 −1   4 T   0 −1   0 −1 −1 −1 −1 −2 −2 −1 −1−1 −1 −2 −1   1   5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1   1 −4 −3−2 11 Y −2 −2 −2 −3 −2 −1 −2 −3   2 −1 −1 −2 −1   3 −3 −2 −2   2   7 V  0 −3 −3 −3 −1 −2 −2 −3 −3   3   1 −2   1 −1 −2 −2   0 −3 −1   4

The percent identity is then calculated as:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}} \right. \\{\quad {{number}\quad {of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}}} \\\left. {{sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

Sequence identity of polynucleotide molecules is determined by similarmethods using a ratio as disclosed above.

Substantially homologous proteins and polypeptides are characterized ashaving one or more amino acid substitutions, deletions or additions.These changes are preferably of a minor nature, that is conservativeamino acid substitutions (see Table 2) and other substitutions that donot significantly affect the folding or activity of the protein orpolypeptide; small deletions, typically of one to about 30 amino acids;and small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue, a small linker peptide of up to about20-25 residues, or a small extension that facilitates purification, (anaffinity tag), such as a poly-histidine tract, protein A (Nilsson etal., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3,1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988),maltose binding protein (Kellerman and Ferenci, Methods Enzymol.90:459-463, 1982; Guan et al., Gene 67:21-30, 1987), or other antigenicepitope or binding domain. See, in general Ford et al., ProteinExpression and Purification 2: 95-107, 1991, which is incorporatedherein by reference. DNAs encoding affinity tags are available fromcommercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.; NewEngland Biolabs, Beverly, Mass.).

TABLE 2 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

The proteins of the present invention can also comprise non-naturallyoccuring amino acid residues. Non-naturally occuring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are knownin the art for incorporating non-naturally occuring amino acid residuesinto proteins. For example, an in vitro system can be employed whereinnonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is carried out in a cell freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science259:806-809, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145-10149, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-19998, 1996) Within a third method, E. coli cells are culturedin the absence of a natural amino acid that is to be replaced (e.g.,phenylalanine). and in the presence of the desired non-naturallyoccuring amino acid(s) (eg., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccuring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occuring amino acid residues can be converted to non-naturallyoccuring species by in vitro chemical modification. Chemicalmodification can be combined with site-directed mutagenesis to furtherexpand the range of substitutions (Wynn and Richards, Protein Sci.2:395-403, 1993).

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad.Sci. USA 88:4498-4502, 1991). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (e.g.,proliferation of islet or β-cells) to identify amino acid residues thatare critical to the activity of the molecule.

Other methods that can be used include phage display (e.g., Lowman etal., Biochem. 30:10832-10837, 1991; Ladner et al., U.S. Pat. No.5,223,409; Huse, WIPO Publication WO 92/06204) and region-directedmutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA7:127, 1988).

Mutagenesis methods as disclosed above can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art canprepare a variety of mature, biologically active polypeptides that arederived from polynucleotides that are substantially homologous tonucleotides 76 to 417 of SEQ ID NO: 1 or allelic variants thereof andretain the properties of the wild-type protein to stimulate isletproliferation, differentiation and/or metabolic processes.

Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryoticcells, particularly culture cells of multicellular organisms, arepreferred. Techniques for manipulating cloned DNA molecules andintroducing exogenous DNA into a variety of host cells are disclosed bySambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, andAusubel et al., ibid., which are incorporated herein by reference.

In general, a DNA sequence encoding a Zins1 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a Zins1 polypeptide into the secretory pathway of a host cell,a secretory signal sequence (also known as a leader sequence, preprosequence or pre sequence) is provided in the expression vector. Thesecretory signal sequence may be that of the Zins1 polypeptide, or maybe derived from another secreted protein (e.g., t-PA) or synthesized denovo. The secretory signal sequence is joined to the Zins1 DNA sequencein the correct reading frame. Secretory signal sequences are commonlypositioned 5′ to the DNA sequence encoding the polypeptide of interest,although certain signal sequences may be positioned elsewhere in the DNAsequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743;Holland et al., U.S. Pat. No. 5,143,830).

If Zins1 polypeptide is expressed in a non-endocrine ornon-neuroendocrine cell, the expression host cell generally will notexpress the prohormone convertases PC2 and PC3, which are believed to beinvolved in the regulated secretory pathway. Another member of thisendoprotease family, furin, is present in most cells and is believed tobe involved in the constitutive secretory pathway. F. Vollenweider etal. have described the role of these prohormone conversion endoproteasesin general, and specifically describe studies involving co-transfectionof COS cells with proinsulin and one of the endoproteases (Diabetes44:1075-80, 1995). Their results showed that PC3 and furin were able tocleave proinsulin at both its junctions; PC2 did not exhibit prohormonecleavage to any significant extent. Without co-transfection of anendoprotease, the prohormone was not converted to any great extent byCOS cells. However, the co-transfection system described is still not anexact model of the natural β cell environment, since β cells make bothPC2 and PC3. Also, a non-endocrine cell does not represent a nativeenvironment for PC2 and PC3 expression. In addition, co-transfection mayresult in general or local overexpression of PC2 and/or PC3, relative tothe native β cell environment. In a preferred embodiment, the host cellswill be co-transfected with a second DNA expression construct comprisingthe following operably linked elements: a transcription promoter; a DNAsegment encoding an endoprotease; and a transcription terminator,wherein the host cell expresses the DNA segment encoding theendoprotease.

Cultured mammalian cells are preferred hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., eds., Current Protocols in MolecularBiology, John Wiley and Sons, Inc., NY, 1987), and liposome-mediatedtransfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al.,Focus 15:80, 1993), which are incorporated herein by reference. Theproduction of recombinant polypeptides in cultured mammalian cells isdisclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339;Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No.4,579,821; and Ringold, U.S. Pat. No. 4,656,134, which are incorporatedherein by reference. Preferred cultured mammalian cells include theCOS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Rockville, Md. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978, which are incorporated herein byreference) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

Other higher eukaryotic cells can also be used as hosts, includinginsect cells, plant cells and avian cells. Transformation of insectcells and production of foreign polypeptides therein is disclosed byGuarino et al., U.S. Pat. No. 5,162,222; Bang et al., U.S. Pat. No.4,775,624; and WIPO publication WO 94/06463, which are incorporatedherein by reference. The use of Agrobacterium rhizogenes as a vector forexpressing genes in plant cells has been reviewed by Sinkar et al., J.Biosci. (Bangalore) 1:47-58, 1987.

Fungal cells, including yeast cells, and particularly cells of the genusSaccharomyces, can also be used within the present invention, such asfor producing Zins1 fragments or polypeptide fusions. Methods fortransforming yeast cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake,U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; andMurray et al., U.S. Pat. No. 4,845,075, which are incorporated herein byreference. Transformed cells are selected by phenotype determined by theselectable marker, commonly drug resistance or the ability to grow inthe absence of a particular nutrient (e.g. leucine). A preferred vectorsystem for use in yeast is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Suitable promoters andterminators for use in yeast include those from glycolytic enzyme genes(see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S.Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092, which areincorporated herein by reference) and alcohol dehydrogenase genes. Seealso U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454, whichare incorporated herein by reference. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia guillermondii and Candida maltosa are known in the art.See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349,which is incorporated herein by reference. Methods for transformingAcremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No.5,162,228, which is incorporated herein by reference. Methods fortransforming Neurospora are disclosed by Lambowitz, U.S. Pat. No.4,486,533, which is incorporated herein by reference.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell.

Zins1 polypeptides can also be used to prepare antibodies thatspecifically bind to Zins1 epitopes, peptides or polypeptides. Methodsfor preparing polyclonal and monoclonal antibodies are well known in theart (see, for example, Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y. 1989; and Hurrell, J.G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Inc., Boca Raton, Fla., 1982, which areincorporated herein by reference). As would be evident to one ofordinary skill in the art, polyclonal antibodies can be generated from avariety of warm-blooded animals, such as humans, horses, cows, goats,sheep, dogs, chickens, rabbits, mice and rats.

The immunogenicity of a Zins1 polypeptide may be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of Zins1 polypeptidesor a portion thereof with an immunoglobulin polypeptide or with amaltose binding protein. The polypeptide immunogen may be a full-lengthmolecule or a portion thereof. If the polypeptide portion is“hapten-like”, such a portion may be advantageously joined or linked toa macromolecular carrier (such as keyhole limpet hemocyanin (KLH),bovine serum albumin (BSA) or tetanus toxoid) for immunization.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingonly non-human CDRs onto human framework and constant regions, orincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Alternative techniques forgenerating or selecting antibodies useful herein include in vitroexposure of lymphocytes to Zins1 polypeptides or peptides, and selectionof antibody display libraries in phage or similar vectors (for instance,through use of immobilized or labeled Zins1 polypeptide or peptide).

Antibodies are defined to be specifically binding if they bind to aZins1 polypeptide with a binding affinity (K_(a)) of 10⁶ M⁻¹ or greater,preferably 10⁷ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ or greater.The binding affinity of an antibody can be readily determined by one ofordinary skill in the art (for example, by Scatchard analysis).

A variety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to Zins1 polypeptides orpeptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Press,1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutant Zins1polypeptides or peptides.

Antibodies to Zins1 polypeptides may be used for tagging cells thatexpress Zins1 polypeptides; for isolating Zins1 polypeptides by affinitypurification; for diagnostic assays for determining circulating levelsof Zins1 polypeptides; for detecting or quantitating soluble Zins1polypeptides as marker of underlying pathology or disease; in analyticalmethods employing FACS; for screening expression libraries; forgenerating anti-idiotypic antibodies; for localization byimmunocytochemistry; and as neutralizing antibodies. Suitable directtags or labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent markers, chemiluminescent markers, magneticparticles and the like; indirect tags or labels may feature use ofbiotin-avidin or other complement/anticomplement pairs as intermediates.Antibodies herein may also be directly or indirectly conjugated todrugs, toxins, radionuclides and the like, and these conjugates used forin vivo diagnostic or therapeutic applications.

Zins1 polypeptide prepared according to the present invention ispurified using methods generally known in the art, such as affinitypurification and separations based on size, charge, solubility and otherproperties of the protein. When the protein is produced in culturedmammalian cells, it is preferred to culture the cells in a serum-freeculture medium in order to limit the amount of contaminating protein.The medium is harvested and fractionated. Preferred methods offractionation include affinity chromatography, Q-Fast Flow Sepharose,MonoQ resin, FPLC, phenyl Sepharose, hydroxyapatite, Mono S and/orS-Sepharose.

Molecules of the present invention can be used to identify and isolatereceptors for Zins1. For example, proteins and peptides of the presentinvention can be immobilized on a column and membrane preparations runover the column (Immobilized Affinity Ligand Techniques, Hermanson etal., eds., Academic Press, San Diego, Calif., 1992, pp.195-202).Proteins and peptides can also be radiolabeled (Methods in Enzymol.,vol. 182, “Guide to Protein Purification”, M. Deutscher, ed., Acad.Press, San Diego, 1990, 721-737) or photoaffinity labeled (Brunner etal., Ann. Rev. Biochem. 62:483-514, 1993. and Fedan et al., Biochem.Pharmacol. 33:1167-1180, 1984) and specific cell-surface proteins can beidentified.

Antibodies to Zins1 proteins and peptides may be used for affinitypurification, for diagnostic assays, for determining circulating levelsof Zins1 polypeptides and as antagonists to block Zins1 binding andsignal transduction in vivo and in vitro.

Proteins of the present invention are useful for stimulatingproliferation or differentiation of pancreatic islets and theircomponent cells which include α-cell, β-cells and δ-cells. Proliferationand differentiation can be measured in vitro using cultured cells or invivo by administering molecules of the claimed invention to theappropriate animal model. For instance, Zins1 transfected orZins1-endoprotease co-transtected expression host cells may be embeddedin an alginate environment and. injected (implanted) into recipientanimals. Alginate-poly-L-lysine microencapsulation, permselective,membrane encapsulation and diffusion chambers have been described as ameans to entrap transfected mammalian cells or primary mammalian cells.These types of non-immunogenic “encapsulations” or microenvironmentspermit the transfer of nutrients into the microenvironment, and alsopermit the diffusion of proteins and other macromolecules secreted orreleased by the captured cells across the environmental barrier to therecipient animal. Most importantly, the capsules or microenvironmentsmask and shield the foreign, embedded cells from the recipient animal'simmune response. Such microenvironments. can extend the life of theinjected cells from a few hours or days (naked cells) to several weeks(embedded cells). The alginate threads, described herein provide asimple and quick means for generating embedded cells. The materialsneeded to generate the alginate threads are readily available andrelatively inexpensive. Once made, the alginate threads are relativelystrong and durable, both in vitro and, based on data obtained using thethreads, in vivo. The alginate threads are easily manipulatable and themethodology is scalable for preparation of numerous threads.

Molecules of the present invention are useful as a reagent for in vitroculturing of islets, and hence their component cells which includeα-cell, β-cells and δ-cells, in vitro, which have been difficult togrow. Cultured islets provide islet cells for transplantation, analternative to whole pancreas transplantation. Assays measuring cellproliferation or differentiation are well known in the art. For example,assays measuring proliferation include such assays as chemosensitivityto neutral red dye (Cavanaugh et al., Investigational New Drugs8:347-354, 1990, incorporated herein by reference), incorporation ofradiolabelled nucleotides (Cook et al., Analytical Biochem. 179:1-7,1989, incorporated herein by reference), incorporation of5-bromo-2′-deoxyuridine (BrdU) in the DNA of proliferating cells(Porstmann et al., J. Immunol. Methods 82:169-179, 1985, incorporatedherein by reference), and use of tetrazolium salts (Mosmann, J. Immunol.Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988;Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., CancerRes. 48:4827-4833, 1988; all incorporated herein by reference). Assaysmeasuring differentiation include, for example, measuring cell-surfacemarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv.Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporatedherein by reference).

Zins1 may also have other insulin-like activities, affecting glucose andlipid metabolism. Assays to measure other cellular responses, thatinclude chemotaxis, adhesion, changes in ion channel influx, regulationof second messenger levels and neurotransmitter release are well knownin the art. See, for example, in “Basic & Clinical Endocrinology Ser.,Vol. Vol. 3,” Cytochemical Bioassays: Techniques & Applications, Chayen;Chayen, Bitensky, eds., Dekker, N.Y., 1983.

Treatment of diabetes using Zins1 will be particularly useful forgestational and Type II (NIDDM) diabetes. In gestational and Type IIdiabetes, the disease is characterized by defects in both insulin action(also referred to as insulin resistance) and insulin secretion. In somepatients, the use of Zins1 alone may be sufficient to eliminate therequirement for exogenous insulin or insulin secretagogues (oralhypoglycemic agents). Zins1 may be used in conjunction with insulin,with insulin sensitizing agents, and oral hypoglycemic agents or withcombinations thereof. Troglitazone is an example of an insulinsensitizing agent. In an exemplary Zins1-insulin sensitizer combinedtreatment, the recipient's insulin resistance is reduced, therebydecreasing the insulin secretion demand, and insulin secretion capacityis enhanced by increases in β-cell mass. Such a treatment provides aβ-cell reserve and results in effective treatment for gestational andType II diabetes. Zins1 may provide treatment for Type I diabetes, iftreatment includes suppression of autoantigenic destruction of β-cellsonce they are stimulated to proliferate and increase in function.

For pharmaceutical use, the proteins of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. Insulin formulations areknown in the art and can provide guidance for molecules of the presentinvention. Intravenous administration will be by bolus injection orinfusion over a typical period of one to several hours. In general,pharmaceutical formulations will include a mature Zins1 protein incombination with a pharmaceutically acceptable vehicle, such as saline,buffered saline, 5% dextrose in water or the like. Formulations mayfurther include one or more excipients, preservatives, solubilizers,buffering agents, albumin to prevent protein loss on vial surfaces, etc.Methods of formulation are well known in the art and are disclosed, forexample, in Remington's Pharmaceutical Sciences, Gennaro, ed., MackPublishihg Co., Easton Pa., 1990, which is incorporated herein byreference. Therapeutic doses will generally be in the range of 0.1 to100 μg/kg of patient weight per day, preferably 0.5-20 μg/kg per day,with the exact dose determined by the clinician according to acceptedstandards, taking into account the nature and severity of the conditionto be treated, patient traits, etc. Determination of dose is within thelevel of ordinary skill in the art. The proteins may be administered foracute treatment, over one week or less, often over a period of one tothree days. In the treatment of diabetes, the molecules of the presentinvention would be used in chronic treatment, over several months oryears. In gestational diabetes, chronic administration would generallybe for weeks. In general, a therapeutically effective amount of Zins1 isan amount sufficient to produce a clinically significant change ininsulin secretory capacity. In a patient, insulin secretory capacity isdetermined by fasting plasma glucose levels or glucose tolerance.Generally, fasting plasma glucose levels equal to, or more than, 126mg/dl indicate diabetes. Impaired glucose tolerance is diagnosed when2-hour plasma glucose levels from a oral glucose tolerance tests aregreater than, or equal to, 140 mg/dl, but less than 200 mg/dl. Above 200mg/dl, diabetes is diagnosed. Generally, treatment would begin whenfasting plasma glucose levels are above 126 mg/dl. Normal plasma glucoselevels are 115 mg/dl, according to standards set by the AmericanDiabetes Association.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Expression of a Biolocically Active Zins1

The Zins1 cDNA was isolated from a human placental library using PCR anddesignated Zins1. The Zins1 cDNA sequence was prepared in a mammalianexpression vector with either a N-terminal or C-terminal poly-His tag.The mammalian expression vector was modified from a vector designatedpHZ-200. pHZ-200 was derived from a mammalian expression vectordesignated pHZ-1 with the only modification being that the dihydrofolatereductase sequence was substituted for the neomycin resistance gene.Plasmid pHZ-1 is an expression vector used to express protein inmammalian cells or in a frog oocyte translation system from mRNAs thathave been transcribed in vitro. The pHZ-1 expression unit comprises themouse metallothionein-1 promoter, the bacteriophage T7 promoter flankedby multiple cloning. banks containing unique restriction sites forinsertion of coding sequences, the human growth hormone terminator andthe bacteriophage T7 terminator. In addition, pHZ-1 contains an E. coliorigin of replication; a bacterial beta lactamase gene; a mammalianselectable marker expression unit comprising the SV40 promoter andorigin, a neomycin resistance gene and the SV40 transcriptionterminator.

The vector used for the N-terminal His tagged Zins1, was designatedpOZ2, and contained at the 5′ end of the cloning site, a tPA leaderfollowed by six histidine residues and a four amino acid spacer (GGSG),as shown in SEQ ID NO: 4 from amino acid residue 36 to residue 45. Thefinal Ser, Gly residues of the spacer constitute a BspE1 restrictionsite, allowing for insertion of the desired cDNA with no extra residues.The downstream 3′ cloning site was Xho1. The zins1 sequence was insertedinto the BspE1/Xho1 site directionally with the predicted mature end ofthe protein at the 5′ end amino acid residue 26 (Ala) of SEQ ID NO: 2.The Xho site occurs directly after an in-frame stop codon. Thisconstruct was designated Zins1pOZ2.

The C-terminal His tagged Zins1 construct was made in pOZ1. This vectoris pHZ-200 based with a Kpn1 site at the 5′ end of the cloning site. Atthe 3′ end the vector contains an in-frame spacer (GSGG) followed by sixhistidine residues. The first two residues of the spacer (GS) constitutea BamHI site which allows for insertion of the cDNA of interest with noextra residues. The zins1 sequence, containing the native leadersequence, was inserted directionally using the Kpn1/BamHI site. A stopcodon occurs after the final His residue. This construct was designatedZins1pOZ1.

The zins1 cDNAs were each co-transfected into BHK 570 cells (ATCCaccession no. 10314) along with cDNA encoding one of two differentconversion endoproteases known to be involved in prohormone processing.These two enzymes, PC2 and PC3, have been shown to be restricted toendocrine and neuroendocrine tissues and cells, with PC3 resulting inmore extensive processing of insulin from its prohormone to active form.

A total of four transfections were performed:

1) zins1pOZ1/PC2 (called zins1C/PC2)

2) zins1pOZ1/PC3 (called zins1C/PC3)

3) zins1pOZ2/PC2 (called zins1N/PC2) and

4) zins1pOZ2/PC3 (called zins1N/PC3).

10 μg of each DNA prep (20 μg total) was ransfected into subconfluentBHK570 cells using ipofectamine reagent (GIBCO-BRL, Gaithersburg, Md.),ccording to the manufacturer's specifications. The following day thetransfected cells were trypsinized, and split at several dilutions up to1:160. The medium was replaced with growth media (Table 3) containingboth 1 μM methotrexate (MTX) and 1+ G418 (neomycin). The pOZ plasmidscontain the DHFR gene conferring resistance to MTX and the plasmidcontaining the PC enzyme contains the neomycin resistance gene. Afterseveral weeks, transfectant pools that had been cultured in 1 μMMTX+G418, and approximately 12 individual clones from each transfectionwere selected for further analyses.

Serum-free conditioned media (Table 4) from each of the pools and cloneswere analyzed for reactivity with an antibody made to a zins1/MBP fusionprotein. The recombinant zinsi protein was affinity purified from thespent culture media using Ni-NTA agarose (Qiagen, Chatsworth, Calif.).Purification was done using a batch process, where 200 μl of Ni resinwas added to 5 ml of conditioned media and incubated overnight on arocking platform at 4° C. The resin was washed 2 times and the proteinseluted directly into 2× tricine gel sample buffer. The samples wereelectrophoresed on 16% Novex tricine gels and blotted ontonitrocellulose. The blots were incubated overnight in a 1:2000 (0.5μg/ml) dilution of the fusion protein antibody.

The blots revealed a broad band of immunoreactivity between 10-18 kDaunder reducing conditions, with distinct bands at 3 and 6 kDa in somelanes. Several clones were picked for further analysis; zins1N/PC3#4,zins1N/PC3#3, zins1N/PC3#9, zins1N/PC2#1, zins1C/PC2#2, zins1C/PC3#9,and zins1C/PC3#1.

N-terminal amino acid sequencing was performed on several clones. Twobands were sequenced from zins1N/PC2 #1. The lower band (^(˜)3 kDa) wasfound to have the sequence SGRHRFDPFXXEVIXDDGTSVKL (amino acid residues115 to 123 of SEQ ID NO: 2, wherein X is Cys), representing the A chainof the molecule. A band slightly above this (^(˜)6 kDa) was sequencedand found to be SQEIHAEFQRGRRHHHHHGGSGAELRGXG (amino acid residues 23 to52 of SEQ ID NO: 4, wherein X is Cys). The first 13 residues are part ofthe tPA leader that was not removed in processing.

Several bands were also sequenced from zins1C/PC3#9 (this moleculecontained the native leader and a C-terminal His tag). Bands of 14.5,9.0, 8.0 and 3.5 kDa were sequenced. The first three bands all startedwith AELRGCG (amino acid residues 26 to 32 of SEQ ID NO: 2), whichappeared to be the N-terminus of the mature zins1 protein (B-chain). The3.5 kDa band started with SGRHRFD (amino acid residues 115 to 121 of SEQID NO: 2), representing the N-terminus of the A-chain.

It was believed that a tag on the N-terminus of the B-chain was lesslikely to interfere with bioactivity than one on the C-terminus of theA-chain. Two clones with relatively high expression of processed proteinwere chosen for use in the alginate threads assay (see Example 2). Thesetwo cell lines, zins1N/PC3#3 and zins1N/PC2#1, differ only by the enzymeco-transfected for prohormone processing. The A-chain produced by thesetwo lines was identical. The B-chain of zins1N/PC2#1 was sequenceanalyzed and contained some of the tPA leader. Zins1N/PC3#3 demonstratedbiological activity in the alginate threads assay (as described inExample 2), and zins1N/PC2#1 did not, suggesting that zins1N/PC3#3 wasproperly processed in this in vivo assay.

TABLE 3 Growth Medium 500 ml Delbecco's Modified Eagle's Medium (DMEM)(Gibco BRL) 5% fetal calf serum (Hyclone, Logan, UT) (1 mM) sodiumpyruvate (Irvine, Santa Ana, CA) (.29 mg/ml) L-glutamine (Hazelton,Lenexa, KS) 1x PSN (5 mg/ml penicillin, 5 mg/ml streptomycin, 10 mg/mlneomycin) (Gibco BRL, Gaithersburg, MD)

TABLE 4 Serum-free Medium 500 ml Dulbecco's Modified Eagle's Medium(DMEM; Gibco BRL) (1 mM) sodium pyruvate (Irvine, Santa Ana, CA) (.29mg/ml) L-glutamine (Hazelton, Lenexa, KS) (1 mg/ml) vitamin K (Merck,Whitehouse Station, NJ) (10 mg/ml) transferrin (JRH, Lenexa, KS) (5mg/ml) fetuin (Aldrich, Milwaukee, WI) (2 ug/ml) selenium (Aldrich,Milwaukee, WI)

Example 2 In Vivo Testing of Zins1 (Zins1) A. Xenogeneic CellTransplantation of Zins1 Gene

i. Preparation of Zins1 Alginate Threads

Briefly, 3% alginate was prepared in USP for injection sterile H₂O(several hours on a rotary shaker at R.T., to get the alginate intosolution), and sterile filtered using an 0.8 μM filter flask (again,several hours to achieve filtration). Just prior to preparation ofalginate threads, the alginate solution was again filtered through a0.45 μM syringe tip filter.

A suspension (containing about 10⁶ to about 10⁸ cells/ml) was mixed at1:1 vol/vol with the 3% alginate solution. One ml of this alginate/cellsuspension was extruded from a 1 cc syringe through a 30 g needle into a100 mM CaCl₂ solution (sterile filtered through a 0.22 μM filter),forming a “thread”. The extruded thread was incubated for about 15 minin the 100 mM CaCl₂ solution; then transferred into a solution of 50 mMCaCl₂; and then into a solution of 25 mM CaCl₂. The thread was thenrinsed with deionized water before incubation in Latctated Ringer'sSolution until the time of injection. Finally, the thread in LactatedRinger's Solution was drawn into a 3 cc syringe barrel (without needleattached). A large bore needle (16 g) was then attached to the syringe,and the thread was intraperitoneally injected into a recipient mouse in^(˜)1.5 ml total volume of the Lactated Ringer's Solution.

In one study, each member of a group (containing six female, one yearold BALB/c mice) was injected with a thread containing either 1×10⁶ wildtype (untransfected) BHK cells; 2×10⁶ zins1N/PC3#3 co-transfected cells;or 4×10⁶ zins1N/PC2#1 co-transfected cells. Blood was drawn at days 12and 15 (non-fasted), and at day 19 (fasted), and serum glucose levels(days 12, 15 and 19) and serum insulin levels (days 12 and 15) weredetermined, as well as cell counts, complete blood chemistries andcomplete blood counts (CBCs). The animals that received the zins1N/PC3#3threads showed lower serum glucose levels at days 12 and 19 than the wtBHK and zins1N/PC2#1 threads-injected animals. At day 12, thezins1N/PC3#3 threads-injected animals showed elevated serum insulinlevels, as compared to the other two groups. Among all of the groups ofanimals, CBCs were comparable.

In a second study, 7 and 6 female BALB/c mice (female, 9 weeks old) wereintraperitoneally injected at day 0 with threads containing about 3×10⁷untransfected BHK cells or threads containing about 5×10⁶ zins1N/PC3#3co-transfected cells, respectively. Another control group of 3 animalsreceived no treatment.

All of the animals were fasted prior to being bled on days −3, 8, 12 and27. For fasting, food was removed at the end of the previous day's lightcycle. The animals experienced a dark cycle without food, and then theanimals were bled after the beginning of the next light cycle.Thereafter, food was restored. At days 8 and 12, the animals that wereinjected with zins1N/PC3#3 threads exhibited a significant decrease inserum glucose (35 and 48 mg/dl, respectively), as compared to animalsthat were injected with wild type BHK cells (65 and 90 mg/dl,respectively). Serum glucose was determined using serum obtained fromwhole blood collected in non-heparinized tubes. The blood wascentrifuged immediately and the serum was analyzed for glucoseconcentration. Serum triglyceride levels were also significantly higherat days 8 and 12 in the animals that were injected with zins1N/PC3#3threads (91 and 60 mg/dl, respectively), as compared to animals thatwere injected with wild type BHK cells (42 and 23 mg/dl, respectively).The zins1N/PC3#3 threads-injected animals exhibited body weights andserum cholesterol levels comparable to those of the wild type BHKthreads-injected animals, and did not appear or behave differently fromthe wild type BHK threads-injected animals.

In a third study, 8 month old db/db mice (very obese, severely diabetic)were injected with wild type BHK threads containing 4×10⁷ cells (n=7) orwith zins1N/PC3#3 threads containing 4×10⁷ cells (n=6). Non-fastedanimals were bled on days −4, 7, 13 and 17. At day 13, blood ureanitrogen levels (an indicator of kidney function) were lower in theanimals that received zins1N/PC3#3 threads, as compared to the BHKthreads control group.

ii. Histology and Histomorphometry

The pancreas and spleen, a portion of the small intestine, omentum andany omental fat that might include pancreas were collected from 15 mice.

The tissues were fixed in 10% NBF (neutral buffered formalin; Surgipath,Richmond, Ill.) overnight. The pancreatic lobes were pressed togetherslightly to expose the largest pancreatic area to make every lobe of thepancreas flatten.

The tissue was dehydrated with a graded series of ethyl alcohols,cleared with xylenc, and infiltrated with PARAPLAST X-TRA (FisherScientific, Pittsburgh, Pa.) using a TISSUE-TEK VIP2000 (Miles, Inc.,Elkhart, Ill.).

The flattened pancreas was removed from the biopsy bag using forceps andembedded longitudinally with PARAPLAST X-TRA. All pancreata wereoriented the same way in the block, with the head of the pancreas placedin one corner of the embedding mold, the tail of the pancreas in theopposite corner, and the body in the middle of the mold.

Each section was trimmed with a Jung Biocut 2035 microtome (Bartels andStout, Inc., Bellevue, Wash.) until the largest pancreatic profile areawas exposed. Sections were cut at 3 μm in thickness.

The sections were stained with Harris hematoxylin (Sigma, St. Louis,Mo.) and Eosin histology staining (Surgipath, Richmond, Ill.). Thenumber and size of islets per longitudinal section of the pancreas werecounted and measured by using a camara-lucida attached to a lightmicroscope (10× objective, Olympus, BH-2), interfaced to a BIOQUANTSYSTEM IV image analysis system (B&M Biometric, Inc., Nashville, Tenn.).After calibration, the electronic pen of the digitizer was used tocarefully trace the outline of each islet profile by screening the wholesection of the pancreas. Simultaneously, the data was computed andstored. Data analyses were performed by using ANOVA (GraphPad Software,San Diego, Calif.) followed by unpaired t test.

The results are shown in FIG. 1 and FIG. 2. FIG. 1 illustrates a 50%increase in the number of islets present in samples taken from animalstreated with BHK cells transfected with zins1 over animals treated withuntransfected BHK cells. FIG. 2 illustrates a trend toward increasedsize of islets seen in animals treated with BHK cells transfected withzins1 versus animals treated with untransfected BHK cells.

B. Administration of Zins1 Purified Protein

Purified, zins1, that is produced by co-expressing the protein with PC3,is administered to normal mice to evaluate the effects on blood glucoseand pancreatic islet histomorphometry. The duration of the study is 27days with dosing for 20 days.

Female Balb/c mice, approximately nine weeks old are divided into thefollowing treatment groups.

Group 1: Vehicle (0.1% BSA/PBS), ip, n=10

Group 2: 1 μg zins1/PC3 per mouse (50 μg/kg), ip, n=10

Group 3: 5 μg zins1/PC3 per mouse (250 μg/kg), ip, n=10

Group 4: Untreated, n=10

On day 0, mice are weighed, ear tagged and injected with 0.1 ml of theappropriate treatment solution. Animals are checked daily for behavioraland grooming changes, and body weights are determined weekly.

Labeling with BrdU (Zymed Laboratories, South San Francisco, Calif.),according the manufacturer's specifications is done from days 8-11 andfrom days 17-19 to label islet cells that are dividing in response tozins1.

Animals are bled on day 8 (a non-fasting sample) under ether anesthesiafor clinical chemistry.

Mice are weighed and bled for serum on day 28. At necropsy, on day 28,the pancreas and a piece of gut for BrdU control are collected. Thepancreas is processed for histomorphbmetric analysis of islet size andnumber as described in A.ii., above. In addition, total cells and isletsare analyzed for BrdU incorporation as described in Ellwart et al.,Cytometry 6:513-520, 1985.

Example 3 Purification and Characterization of Zins1 Protein A.Purification of Zins1 Protein

The construct encoding the 124 amino acid Zins1 NF+PC3 (Zins1poZ2/PC3;described in Ex. 1) expressed in BHK cells was purified by affinitychromatography on anti-flag Sepharose (Eastman Kodak, Rochester, N.Y.),according to the manufacturer's specifications. Antigen was eluted withflag peptide, and further purified by gel filtration chromatography onSepharose G-50 (Eastman Kodak). A total of 4.5 mg of Zins1 NF+PC3 waspurified.

Analysis of the purified material by nonreducing SDS-PAGE, followed bystaining with Coomassie Blue, revealed a mixture of at least fourpeptides of apparent molecular weights 14,000-25,000. By staining, eachof the four peptides was present in approximately equimolar amounts andeach of these bands appeared to cross react with anti-Flag antibodiesupon Western blotting. Under reducing conditions, the electrophoreticprofile was altered with each of bands exhibiting somewhat greaterelectrophorectic mobility. In addition, the major Coomassie Blue-stainedprotein observed under these conditions was a protein of ^(˜)4 kDa. Thisband did not cross react with anti-Flag antibodies on Western blots.

Purified Zins 1NF+PC3 was probed on Western blots with each of threeanti-Zins 1 peptide antibodies and anti-Flag antibodies as a control.The peptides used for antibody production in rabbits were:

Zins1-DC-1, LSQLLRESLAAELRG, residues 16 to 30 of SEQ ID NO: 2 (spanningthe putative N-terminus/“B” chain junction);

Zins1-DC-2, LLESGRPKEMVSTSNNKD, residues 57 to 75 of SEQ ID NO: 2 (theamino terminus of the “B” chain/“C” chain junction as predicted byChassin et al., 1995, ibid.);

Zins1-DC-3, LKKIILSRKKRSGRHR, residues 104 to 119 of SEQ ID NO: 2(spanning the putative “C” chain/“A” chain junction)

The anti-Zins 1-DC-1 antibody did not react with any band on reducing ornonreducing SDS-PAGE gels. Since only the five C-terminal residues ofthis peptide (i.e., residues 26-30 of SEQ ID NO: 2) were containedwithin the sequence of Zins1pOZ2/PC3, this indicated that no antibodiesto this sequence were present. The lack of immunoreactivity alsosuggested that Zins1pOZ2/PC3 is correctly flag-tagged, since nocrossreactivity was observed to amino acids N-terminal to the “B” chainjunction.

The anti-Zins1 DC-3 did not react with any peptides on the Westernblots, as well. These antibodies were directed against a peptide thatspans the putative “C” chain/“A” chain junction. These results suggestedthat this region was cleaved during processing of the Zins1, a findingconsistent with the PVDF blotting/sequencing. Lack of immunoreactivitywith larger or smaller bands (unprocessed “B/C+A” or processed “B/C” and“A” chain) suggested that the epitope was at the “C” chain/“A” chainjunction.

Results obtained with the anti-Zins1-DC-2, antibodies directed against apeptide from the putative “C”-peptide, were different. The reactivitylooked identical to that observed with anti-Flag antibodies, namelyreactivity was seen in several bands around ^(˜)20 kDa. These bandsshowed a small decrease in apparent size upon reduction.

B. Characterization of Zins1 Protein

The N-terminally tagged Zins1 protein purified above was characterizedusing N-terminal sequence analysis, glycosidase PAGE analysis,monosaccharide composition analysis and mass spectral analysis.

N-terminal sequence analysis was done as follows:

A sample of Zins 1, purified as described above, was run on a NOVEX 18%Tris-Glycine gel (NOVEX, San Diego, Calif.) under reducing conditions(2-mercaptoethanol). An clectroblot transfer to PVDF membrane wasperformed in 10 mM CAPS buffer pH 11.0, 10% methanol at 200 mA for 1hour at 4° C. The PVDF blot was visualized with Coomassie blue staining.Stained protein bands were excised for Edman degradation N-terminalprotein sequencing on an Applied Biosystems 476A Protein Sequencer(Foster City, Calif.) using standard protocols and FSTBLT cycles. Thedata was analyzed using Applied Biosystems Model 610A Data AnalysisSystem, v. 1.2.2).

Liquid Chromatography—Mass Spectrometry (LCMS) was performed as follows:

A Michrom BioResources MAGIC 2002 HPLC system (Michrom BioResources,Inc., Auburn, Calif.) equipped with a 1.0×150 mm Monitor C18 100 Å 5 mcolumn (Michrom BioResources, Inc.) was used at a flowrate of 50 μl/minand a column temperature of 30° C. Typically, 5.0 μg of whole ordigested protein was injected onto the column equilibrated in 5% B and alinear gradient from 5 to 85% B over 80 minutes was immediatelyinitiated (A: 2% acetonitrile+0.1% acetic acid+0.020% TFA, B: 90%acetonitrile+0.1% acetic acid+0.018% TFA) The outlet from the HPLC UVdetector was plumbed directly into a Finnigan LCQ Ion Trap MassSpectrometer (Thermoquest Corp., San Jose, Calif.) with no flowsplitting, a heated capillary temperature of 220° C., and a sheath gasflow of 75 (arbitrary units). The source voltage was 5.60 kV and thecapillary voltage was 41.00 V. Mass spectra from 300-2000 m/z wererecorded continuously during the gradient with 3 microscans per fullscan. The most intense [M+2H]2+ ion in each spectrum was automaticallyselected by the LCQ for zoom scan and MSMS at 25% collision energy.

As described above, initial SDS-PAGE analysis of the non-reduced,affinity purified Zins1 NF revealed a series of bands between 15-20 kDa.Upon reduction of the protein, this series of bands shifts to 12-18 kDaand a new band appears with an apparent molecular weight of 4 kDa. Whilethe non-reduced 15-20 kDa and reduced 12-18 kDa bands bind anti-FLAGantibody in a Western blot, the reduced 4 kDa band does not. N-terminalsequence analysis was carried out on bands excised from a PVDF blot ofan 18% Tris-Glycine reducing SDS-PAGE gel, specifically, the bands at 4,12, and 18 kDa. The two high molecular weight bands both gave singlesequences beginning at the first residue of the FLAG sequence, Residue 1(Asp; SEQ ID NO: 5), and both continued through the expected N-terminalsequence for Zins1 NF to residue 25 (Leu) of SEQ ID NO: 5 The 4 kDa bandwas >85% single sequence beginning at residue 100 (Ser) and continuingthrough the expected sequence to residue 122 (Leu) of SEQ ID NO: 5. Thesequencing data corroborates the observed pattern in the Western blotwith the upper molecular weight bands containing the FLAG sequence andthe 4 kDa band containing no FLAG sequence. In addition the sequencingdata indicates that Zins1 NF has been processed by the co-expressed PC3at the expected C/A junction, cleaving the protein after the residues96-99 (ArgLysLysArg) of SEQ ID NO: 5, to yield an A chain beginning atresidue 100 (Ser) of SEQ ID NO: 5. Since the heterogeneity observed inthe purified Zins1 NF is not due to differential processing of thepolypeptide chain at the N-terminus or the C/A junction, it may be dueto differential processing at the B/C junction or glycosylation events.

In order to ascertain if glycosylation is a factor in the observedheterogeneity, Zins1 NF was digested with PNGaseF and sialidase.Glycosidase PAGE analysis was performed as follows:

25 μg of protein was subjected to PNGaseF (peptide-N-glycosidase F)digestion. The protein was digested at 0.5 mg/ml protein. and 0.2 U/mlOxford GlycoSystems (Rosedale, N.Y.) recombinant F. meningosepticumPNGaseF in 20 mM sodium phosphate+50 mM EDTA pH 7.5. The digest wasincubated at 37° C. for 24 hrs. 5 μg of the treated protein was thenanalyzed by SDS-PAGE.

25 μg of protein was subjected to sialidase digestion. The protein wasdigested at 0.5 mg/ml protein and 4.0 U/ml Oxford GlycoSystemsrecombinant C. perfringens sialidase in 50 mM sodium acetate pH 5.0. Thedigest was incubated at 37° C. for 24 hrs. 5 μg of the treated proteinwas then analyzed by SDS-PAGE.

5 μg each of untreated, PNGaseF-treated, and sialidase-treated Zins1 NFwas diluted with an equal volume of NOVEX 2× Tris-Glycine SDS samplebuffer (NOVEX, San Diego, Calif.), boiled for 3-5 minutes, and loadedonto a NOVEX 18% Tris-Glycine gel. In addition, 5 μg each of untreated,PNGaseF-treated, and sialidase-treated Zins1 NF was diluted with anequal volume of NOVEX 2× Tris-Glycine SDS sample buffer (NOVEX, SanDiego, Calif.) containing 5% b-mercaptoethanol, boiled for 3-5 minutes,and loaded onto a NOVEX 18% Tris-Glycine gel. Both the non-reduced andreduced gels were run at a constant voltage of 125V and visualized withCoomassie Blue staining. NOVEX Mark 12 Wide Range Protein Standards wereused to determine apparent molecular weights.

Non-reducing and reducing SDS-PAGE analysis of the PNGaseF-treated Zins1NF revealed no differences relative to the untreated material. Treatmentof Zins1 NF with sialidase resulted in a shift of the upper molecularweight bands to 14-18 kDa in the non-reducing gel and 12-16 kDa in thereducing gel, an average loss of ^(˜)1-2 kDa in apparent molecularweight. The band at 4 kDa in the reducing gel was unaffected. Theresults of the glycosidase treatment indicates that the single potentialN-glycosylation site present in Zins1 NF, AsnLeuSer, residues 73-75 ofSEQ ID NO: 5, is not glycosylated. However, the sialidase resultssuggest that Zins1 NF is O-glycosylated with sialylated O-glycans andthat these O-glycans are not located on the A chain.

Confirmation of the putative O-glycosylation was obtained viamonosaccharide composition analysis. Monosaccharide composition forZins1 was analyzed as follows: Monosaccharide composition was carriedout on a Dionex system composed of a DX500 HPLC with an ED40electrochemical detector, a GP40 pump, and a CARBOPAC-PA 10 column(Dionex, Sunnydale, Calif.). In both types of analyses, Dionexmonosaccharide standards were used to calibrate the instrument. Theglycoprotein fetuin was used as a positive control (Sigma. St. Louis,Mo.).

For sialic acid analysis, 2-8 μg of Zins1 NF was vacuum centrifuged todryness without heat and reconstituted in 500 μl of 0.1 N TFA. Aftermixing the samples were incubated at 80° C. for 60 min., vacuumcentrifuged to dryness without heat and reconstituted in 100 μl ofdistilled H₂O. 25 μl of hydrosylate was injected onto the Dionex systemequilibrated in 50 mM sodium acetate/100 mM NaOH. A gradient to 180 mMsodium acetate/100 mM NaOH over 25 minutes was used. Triplicate analyseswere averaged.

For neutral monosaccharide analysis, 2-8 μg of NF-zins1 was vacuumcentrifuged to dryness without heat and reconstituted in 500 μl of 2.0 NTFA. After mixing the samples were incubated at 100° C. for 4 hours,vacuum centrifuged to dryness without heat and reconstituted in 100 μlof distilled H₂O. 25 μl of hydrosylate was injected onto the Dionexsystem equilibrated in 18 mM NaOH. An isocratic separation at 18 mM NaOHover 25 minutes was used. Triplicate analyses were averaged.

Sialic acid composition analysis showed that Zins1 NF has 6.0±0.5 molesof sialic acid per mole of protein and that these sialic acids areN-acetylneuraminic acid (NeuNAc) and not N-glycolylrneuraminic acid(NeuNGc) residues. Neutral monosaccharide composition analysis showedthat NF-zins1 has 3.7±1.0 moles N-acetylgalactosamine (GalNAc) and1.3±0.4 moles galactose (Gal) per mole of protein. These figures areconsistent with an average of 2.7 disialylated mucin-type O-glycans(NeuNAcα2-3Galβ1-3(NeuNAcα2-6)GalNAc-Ser/Thr) on each molecule ofNF-zins1. No N-acetylglucosamine (GlcNAc) or fucose (Fuc) was detectedand only a small amount of mannose (0.4±0.2 moles mannose per moleprotein), consistent with a lack of N-glycans.

LCMS analysis of reduced Zins1 NF resulted in a very broad peak elutingfrom ^(˜)27-34 minutes with a sharp peak superimposed at 30.7 minutes.The broadness of the peak and the dearth of ions generated from it istypical of heterogeneous glycosylated proteins. One mass was discernibleat 33.5 minutes, 12831.7 Da, a mass consistent with that expected foruncleaved B/C chain (Asp1-Arg99, residues 1 to 99 of SEQ ID NO: 5) with2 disialylated mucin type O-glycans, 12834.2 Da. Presumably the materialeluting before this mass is more heavily glycosylated (and thus moreheterogeneous) B/C chain. The peptide eluting at 30.7 minutes ionizedwell and has a mass of 2789.5 Da, consistent with the expected molecularweight (2789.2 Da) of the predicted A chain, Ser100-Thr 124 (residues100 to 124 of SEQ ID NO: 5.

LCMS and concurrent MSMS analysis of trypsinized native Zins1 NFrevealed tryptic peptides from 89% of the complete sequence of Zins1 NFfrom Asp1 to Thr124. The tryptic peptides from the “C peptide” were notany less abundant than those from the “B chain” or A chain, suggestingthat there is no B/C junction processing in Zins1 NF. A mass of 4289.5Da was observed eluting at 29.4 minutes; the mass expected forGly15-Arg19+His23-Lys33+His103-Thr124 (residues of SEQ ID NO: 5) joinedby three disulfide bonds is 4289.0 Da. This observed mass is consistentwith, though not exclusively, the disulfide bonding pattern expectedfrom homology to the insulin family, i.e. Cys16-Cys110, Cys28-Cys123,and Cys109-Cys114 as shown in SEQ ID NO: 5. Furthermore, masses wereobserved that are consistent with tryptic peptide Thr34-Lys50 (as shownin SEQ ID NO: 5)+0-1 O-glycans (27.6 minutes), Glu51-Lys79 (as shown inSEQ ID NO: 5)+2-3 O glycans (29.5 minutes), and Asp60-Lys79 (as shown inSEQ ID NO: 5)+0-1 O-glycans (30.2-31.1 minutes).

The observed pattern of tryptic O-glycopeptides reveals that there are 4O-glycosylation sites in the “C peptide” region of Zins1 NF. One site iscontained in O-glycopeptide Thr34-Lys50 (as shown in SEQ ID NO: 5) andthe modified residue is Thr34, Thr36, Thr37, Thr38, or Ser46 (as shownin SEQ ID NO: 5). Two sites are contained in O-glycopeptide Glu51-Lys59(as shown in SEQ ID NO: 5) and the modified residues are Ser54, Thr55,and/or Ser56 (as shown in SEQ ID NO: 5). Finally, one site is containedin O-glycopeptide Asp60-Lys79 (as shown in SEQ ID NO: 5) and themodified residue is Thr66, Thr67, Ser68, or Ser75 (as shown in SEQ IDNO: 5).

Example 4 In Vitro Testing of Zins1 Protein A. Isolation of PositiveControl for Islet Proliferation Assay

To establish an assay to measure proliferation in islets in vitro, apositive control was isolated and characterized as fetal antigen 1 (FA1)as follows:

Pancreata from four 8-11 week old, p53−/− male mice (Taconic Farms,Germantown, N.Y.) were excised. The dissected pancreata were placed in asterile 30 mm petri dish containing 7 ml of HBSS (Table 5)+5 mM CaCl₂,and the tissue was minced for exactly 2 minutes. Using a 10 ml pipet,the tissue was transferred to a sterile 25 ml screw-capped, round-bottomcentrifuge tube, and 20 ml HBSS+5 mM CaCl₂, was added. After settling(about 2 minutes), the supernatant (containing fat and connectivetissue) was removed. This procedure was repeated twice.

24 mg collagenase (Collagenase Type XI, Sigma Chemical Co., St. Louis,Mo.) was dissolved in 12 ml HBSS+5 mM CaCl₂ just prior to use, and waskept on ice. The collagenase solution (6 ml) was added to the mincedtissue to a final concentration of 2 mg/ml. The cell mixture was placedon a shaker (300 rpm at 37° C.) for 15 minutes, and then quicklycentrifuged for ^(˜)2 minute at 800 rpm in a Beckman CS-6R centrifugewith a swinging bucket rotor (Beckman Instruments, Palo Alto, Calif.).The supernatant was discarded.

6 ml fresh collagenase solution and 800 μl DNAse were added, and thecell mixture was returned to the shaker for up to 20 minutes. 50 μl ofcell mixture sample was added to 150 μl DTZ (Table 6), and was examinedusing a dissecting microscope to ascertain when the islet cells wereisolated, but not over-digested.

When the islet cells were isolated, the collagenase digestion wasstopped by adding 15 ml HBSS+10% FBS to the mixture, and the mixture wasthen centrifuged in a Beckman CS-6R centrifuge with a swinging bucketrotor (Beckman Instruments, Palo Alto, Calif.) ^(˜)2 minutes at 800 rpm(the “wash step”). The supernatant was removed and discarded. The washstep was repeated two more times.

After washing, the cell pellet was resuspended in 2 ml HBSS, and theresuspended preparation was placed on two PERCOLL (Table 7) gradients (3ml 40% PERCOLL and 3 ml 60% PERCOLL per 50 ml tube). One ml of this cellsuspension was added to each tube. An additional 2 ml of HBSS was usedto sequentially rinse the tubes from which the cell pellets werepreviously removed. This 2 ml of rinse suspension was added in 1 mlaliquots to each of the two gradients. Thus, each 50 ml tube had 2 ml ofcell suspension on the top, then 3 ml of 40% PERCOLL, and finally 3 mlof 60% PERCOLL. The tubes were centrifuged in a Beckman CS-6R centrifugewith a swinging bucket rotor (Beckman Instruments) at 1850 rpm for 20minutes, without the brake on.

After centrifugation, the top and bottom gradient interfaces wereremoved with a sterile transfer pipet, and each interface wastransferred to a separate 50 ml tube. HBSS +10% FBS was added to theinterface and washed by centrifugation in a Beckman CS-6R centrifugewith a swinging bucket rotor (Beckman Instruments) for 10 minutes at 925rpm.

The top and bottom interfaces were filtered through a 70 μm nylon cellstrainer (Becton Dickinson & Co., San Jose, Calif.). The islet cellsremained on the filter, and exocrine tissue passed through. The filterwas flipped upside-down in a 60 mm petri dish, and the islet cells werewashed into the dish. To ensure their isolation from other tissue, theislet cells were plucked into a clean 60 mm non-tissue culture-treateddish containing RPMI growth medium (Table 8) +10% FBS. The islets wereincubated at 37° C., 5% CO₂ and the medium was changed at 24 and 48hours.

TABLE 5 HBSS 50 ml 10X HBSS 10 ml 1M Hepes 2.4 ml 7.5% NaHCO₃ 5 ml PSN(100 × penicillin-streptomycin-neomycin) Add sterile milli-Q water up to500 ml and filter

TABLE 6 DTZ 10 mg DTZ (Sigma, St. Louis, MO) 1 ml DMSO, to dissolve DTZMake to 10 ml final volume with HBSS Filter

TABLE 7 PERCOLL 90% : 90 ml 100% PERCOLL + 10 ml 10X HBSS 60% : 30 ml90% PERCOLL + 15 ml HBSS 40% : 20 ml 90% PERCOLL + 25 ml HBSS

TABLE 8 RPMI 2.4 ml 7.5% NaHCO₃ 10 ml 1M Hepes 5 ml 100 × PSN 5 ml 100 ×Glutamine RPMI-1640 (to a final volume of 450 ml) 50 ml fetal calf serum

Islets, obtained as described above, were placed in a 60 mm petri dishin RPMI +10% FBS, and nine days later the whole islets were removed fromthe petri dish and replated in another 60 mm petri dish. Twenty one dayslater, the first dish was confluent, and the cells were removed withtrypsin and passed into a T₂₅ flask.

Conditioned culture medium removed from these islet cells was added tocultures of normal BALB/c islets were isolated in MATRIGEL BasementMembrane Matrix (Collaborative Biomedical Products, Bedford, Mass.). Thenormal mouse islet phenotype changed, becoming huge with much branchingand forming cyst-like structures. This conditioned medium was designatedIDC53.1. Various other conditioned media obtained either from culturesof osteoclast, osteoblast or dendritic cells obtained from p53−/−knockout mice (see WO 9607733), or from cultures of normal C57/Black 6islet cells, did not exhibit this activity. In addition, normal BALB/cislets placed in this conditioned medium developed “cobblestone” cellsall around the islet. This effect was not seen when various otherconditioned media were tested.

A BrdU incorporation study using BALB/c islets incubated with IDC53.1conditioned medium (CM) was performed, to test whether there were cellswithin the islets that were proliferating. Briefly, one group of fourT_(12.5) flasks (Becton Dickinson) was inoculated with 100 islets each,and 5×IDC53.1 CM+0.5% FBS was added to each flask. Another group ofthree T_(12.5) flasks was inoculated with 100 islets each, and SFIFmedium (serum free/insulin free medium; Becton Dickinson) +0.5% FBS wasadded.

BrdU (Becton Dickinson) was added to the islet cell cultures daily, to afinal concentration of 10 μM. A flask from each group was harvested ondays 4, 8 and 12. On day 8, two of the four flasks in the IDC53.1 CMtest group were harvested. One of these flasks was used for an isotypecontrol. The protocol and reagents for BrdU assay are available fromBecton Dickinson Immunocytometry Systems, San Jose, Calif., and wereused according to the manufacturer's specifications.

For each harvested flask, the islets were harvested, washed twice in 1%BSA/PBS, and centrifuged at 800 rpm for 10 minutes. The pellet wasresuspended in 200 μl of 1×PBS on ice. Islets were slowly added to 2.5ml cold 70% ethanol in a siliconized glass tube while maintaining avortex. The islets were incubated on ice for 30 minutes, and the resultwas fixed islet cells. The fixed islets were centrifuged at 1000 rpm for10 minutes at 10° C., and the ethanol was carefully removed.

One ml of 2 N HCl/Triton X-100 was slowly added to the cells, a fewdrops at a time, while maintaining a vortex. The mixture was incubatedat room temperature for 30 minutes, to denature the DNA and producesingle-stranded molecules. The preparation was centrifuged at 1000 rpmfor 10 minutes, and then the supernatant was removed and the pelletresuspended in 1 ml of 0.1 M Na₂B₄O₇.10 H₂O, pH 8.5, to neutralize theacid. The resultant cells may be stored at this point by centrifuging,resuspending in cold 70% ethanol, and storing at −20° C.

The cells are then centrifuged at 1000 rpm for 10 minutes, washed with 1ml of 0.5% TWEEN 20 in 1% BSA/PBS (TWEEN/BSA/PBS), and resuspended in100 μl TWEEN/BSA/PBS. To this resuspended preparation was added 20 μl ofFITC-labeled anti-BrdU antibody or isotype control. The mixture wasincubated overnight on a shaker at 4° C. for whole islets. Thereafter,the cells were washed 3 times using 1 ml TWEEN/BSA/PBS, where each washwas performed for at least 2 hours on the shaker. Preferable, the finalwash is left overnight.

The islet preparation was then mounted on glass slides with depressionsto prevent the islets from losing their shape. FLUOROGUARD AntifadeReagent (BioRad, Hercules, Calif.) was the mounting medium used. Allpositive BrdU cells per islet were counted for each of the three harvestdays. On Day 4, there were 1.5 times more positive cells in the isletscultured in the 5×IDC53.1 CM than in the control. On Day 8, there were2.9 times more positive cells, and on Day 12 there were 3.5 times morepositive cells, as compared to the control.

Islets were prepared as described above for a BrdU assay, but afterincubation with the BrdU, the islets were harvested, fixed, embedded,sliced and stained for anti-Brdu, anti-insulin, anti-glucagon andanti-somatastatin using standard immunohistochemistry techniques. Thepositive BrdU cells were also positive for insulin, and were negativefor glucagon and somatostatin, strongly suggesting that the cells areβ-cells.

Using standard immunodepletion methods, it was demonstrated that FA1 wasa factor in islet proliferation, and useful as a positive control fortesting islet proliferation.

B. Zins1 Testing in In Vitro Islet Assay

Normal BALB/c islets were isolated from 8.5 week old male mice. Theislets were plated into a 96-well flat bottom plate, with approximately15 islets/well in serum-free/insulin-free +0.5w FCS medium, induplicate. Zins1 diluted serum-free/insulin-free +0.5% FCS medium wasadded at concentrations of 1-20 ng/ml, along with a negative control ofserum-free/insulin-free +0.5% FCS medium, and a positive control ofconditioned medium as described in A.

At day 5, the wells to which positive control and all concentrations ofZins1 had been added, cells were proliferating, with optimal growth inthe 1-10 ng/ml doses. At day 8, the 1-10 ng/ml dose range ofZins1clearly contained adherent cells that appeared to be growing fromthe islets. The cells which grew out of islets treated with Zins1exhibited a spindle morphology in contrast to the FA-1 treated islets,which yielded cobblestone monolayers. Islets treated only with basalmedium had no cell outgrowth and appeared senescent.

These data show that Zins1 can maintain islets in a viable condition andfurther stimulate expansion of specific cell types by outgrowth from theislets.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

7 420 base pairs nucleic acid double linear cDNA Coding Sequence 1...4171 ATG GCC AGC CTG TTC CGG TCC TAT CTG CCA GCA ATC TGG CTG CTG CTG 48 MetAla Ser Leu Phe Arg Ser Tyr Leu Pro Ala Ile Trp Leu Leu Leu 1 5 10 15AGC CAA CTC CTT AGA GAA AGC CTA GCA GCA GAG CTG AGG GGA TGT GGT 96 SerGln Leu Leu Arg Glu Ser Leu Ala Ala Glu Leu Arg Gly Cys Gly 20 25 30 CCCCGA TTT GGA AAA CAC TTG CTG TCA TAT TGC CCC ATG CCT GAG AAG 144 Pro ArgPhe Gly Lys His Leu Leu Ser Tyr Cys Pro Met Pro Glu Lys 35 40 45 ACA TTCACC ACC ACC CCA GGA GGG TGG CTG CTG GAA TCT GGA CGT CCC 192 Thr Phe ThrThr Thr Pro Gly Gly Trp Leu Leu Glu Ser Gly Arg Pro 50 55 60 AAA GAA ATGGTG TCA ACC TCC AAC AAC AAA GAT GGA CAA GCC TTA GGT 240 Lys Glu Met ValSer Thr Ser Asn Asn Lys Asp Gly Gln Ala Leu Gly 65 70 75 80 ACG ACA TCAGAA TTC ATT CCT AAT TTG TCA CCA GAG CTG AAG AAA CCA 288 Thr Thr Ser GluPhe Ile Pro Asn Leu Ser Pro Glu Leu Lys Lys Pro 85 90 95 CTG TCT GAA GGGCAG CCA TCA TTG AAG AAA ATA ATA CTT TCC CGC AAA 336 Leu Ser Glu Gly GlnPro Ser Leu Lys Lys Ile Ile Leu Ser Arg Lys 100 105 110 AAG AGA AGT GGACGT CAC AGA TTT GAT CCA TTC TGT TGT GAA GTA ATT 384 Lys Arg Ser Gly ArgHis Arg Phe Asp Pro Phe Cys Cys Glu Val Ile 115 120 125 TGT GAC GAT GGAACT TCA GTT AAA TTA TGT ACA TAG 420 Cys Asp Asp Gly Thr Ser Val Lys LeuCys Thr 130 135 139 amino acids amino acid single linear proteininternal 2 Met Ala Ser Leu Phe Arg Ser Tyr Leu Pro Ala Ile Trp Leu LeuLeu 1 5 10 15 Ser Gln Leu Leu Arg Glu Ser Leu Ala Ala Glu Leu Arg GlyCys Gly 20 25 30 Pro Arg Phe Gly Lys His Leu Leu Ser Tyr Cys Pro Met ProGlu Lys 35 40 45 Thr Phe Thr Thr Thr Pro Gly Gly Trp Leu Leu Glu Ser GlyArg Pro 50 55 60 Lys Glu Met Val Ser Thr Ser Asn Asn Lys Asp Gly Gln AlaLeu Gly 65 70 75 80 Thr Thr Ser Glu Phe Ile Pro Asn Leu Ser Pro Glu LeuLys Lys Pro 85 90 95 Leu Ser Glu Gly Gln Pro Ser Leu Lys Lys Ile Ile LeuSer Arg Lys 100 105 110 Lys Arg Ser Gly Arg His Arg Phe Asp Pro Phe CysCys Glu Val Ile 115 120 125 Cys Asp Asp Gly Thr Ser Val Lys Leu Cys Thr130 135 480 base pairs nucleic acid double linear cDNA Coding Sequence1...477 3 ATG GAT GCA ATG AAG AGA GGG CTC TGC TGT GTG CTG CTG CTG TGTGGC 48 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 15 10 15 GCC GTC TTC GTT TCG CCC AGC CAG GAA ATC CAT GCC GAG TTC CAG AGA96 Ala Val Phe Val Ser Pro Ser Gln Glu Ile His Ala Glu Phe Gln Arg 20 2530 GGA CGC AGA CAT CAC CAT CAC CAT CAC GGT GGC TCC GGA GCA GAG CTG 144Gly Arg Arg His His His His His His Gly Gly Ser Gly Ala Glu Leu 35 40 45AGG GGA TGT GGT CCC CGA TTT GGA AAA CAC TTG CTG TCA TAT TGC CCC 192 ArgGly Cys Gly Pro Arg Phe Gly Lys His Leu Leu Ser Tyr Cys Pro 50 55 60 ATGCCT GAG AAG ACA TTC ACC ACC ACC CCA GGA GGG TGG CTG CTG GAA 240 Met ProGlu Lys Thr Phe Thr Thr Thr Pro Gly Gly Trp Leu Leu Glu 65 70 75 80 TCTGGA CGT CCC AAA GAA ATG GTG TCA ACC TCC AAC AAC AAA GAT GGA 288 Ser GlyArg Pro Lys Glu Met Val Ser Thr Ser Asn Asn Lys Asp Gly 85 90 95 CAA GCCTTA GGT ACG ACA TCA GAA TTC ATT CCT AAT TTG TCA CCA GAG 336 Gln Ala LeuGly Thr Thr Ser Glu Phe Ile Pro Asn Leu Ser Pro Glu 100 105 110 CTG AAGAAA CCA CTG TCT GAA GGG CAG CCA TCA TTG AAG AAA ATA ATA 384 Leu Lys LysPro Leu Ser Glu Gly Gln Pro Ser Leu Lys Lys Ile Ile 115 120 125 CTT TCCCGC AAA AAG AGA AGT GGA CGT CAC AGA TTT GAT CCA TTC TGT 432 Leu Ser ArgLys Lys Arg Ser Gly Arg His Arg Phe Asp Pro Phe Cys 130 135 140 TGT GAAGTA ATT TGT GAC GAT GGA ACT TCA GTT AAA TTA TGT ACA TAG 480 Cys Glu ValIle Cys Asp Asp Gly Thr Ser Val Lys Leu Cys Thr 145 150 155 159 aminoacids amino acid single linear protein internal 4 Met Asp Ala Met LysArg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe ValSer Pro Ser Gln Glu Ile His Ala Glu Phe Gln Arg 20 25 30 Gly Arg Arg HisHis His His His His Gly Gly Ser Gly Ala Glu Leu 35 40 45 Arg Gly Cys GlyPro Arg Phe Gly Lys His Leu Leu Ser Tyr Cys Pro 50 55 60 Met Pro Glu LysThr Phe Thr Thr Thr Pro Gly Gly Trp Leu Leu Glu 65 70 75 80 Ser Gly ArgPro Lys Glu Met Val Ser Thr Ser Asn Asn Lys Asp Gly 85 90 95 Gln Ala LeuGly Thr Thr Ser Glu Phe Ile Pro Asn Leu Ser Pro Glu 100 105 110 Leu LysLys Pro Leu Ser Glu Gly Gln Pro Ser Leu Lys Lys Ile Ile 115 120 125 LeuSer Arg Lys Lys Arg Ser Gly Arg His Arg Phe Asp Pro Phe Cys 130 135 140Cys Glu Val Ile Cys Asp Asp Gly Thr Ser Val Lys Leu Cys Thr 145 150 155124 amino acids amino acid single linear 5 Asp Tyr Lys Asp Asp Asp AspLys Gly Ser Ala Glu Leu Arg Gly Cys 1 5 10 15 Gly Pro Arg Phe Gly LysHis Leu Leu Ser Tyr Cys Pro Met Pro Glu 20 25 30 Lys Thr Phe Thr Thr ThrPro Gly Gly Trp Leu Leu Glu Ser Gly Arg 35 40 45 Pro Lys Glu Met Val SerThr Ser Asn Asn Lys Asp Gly Gln Ala Leu 50 55 60 Gly Thr Thr Ser Glu PheIle Pro Asn Leu Ser Pro Glu Leu Lys Lys 65 70 75 80 Pro Leu Ser Glu GlyGln Pro Ser Leu Lys Lys Ile Ile Leu Ser Arg 85 90 95 Lys Lys Arg Ser GlyArg His Arg Phe Asp Pro Phe Cys Cys Glu Val 100 105 110 Ile Cys Asp AspGly Thr Ser Val Lys Leu Cys Thr 115 120 14 amino acids amino acid singlelinear peptide Other 4...13 Xaa is any amino acid except Cys 6 Leu CysGly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 15 amino acidsamino acid single linear peptide Other 3...5 Xaa is any amino acidexcept Cys (A) NAME/KEY Other (B) LOCATION 7...14 (D) OTHER INFORMATIONXaa is any amino acid except Cys 7 Cys Cys Xaa Xaa Xaa Cys Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Cys 1 5 10 15

We claim:
 1. An isolated protein produced by a method comprising:culturing a host cell into which has been introduced a DNA expressionvector comprising the following operably linked elements: atranscription promoter; a DNA segment comprising a nucleotide sequenceshown in SEQ ID NO: 1 from nucleotide 76 to nucleotide 417; and atranscription terminator, wherein said host cell expresses thepolypeptide encoded by said DNA segment; and recovering said protein. 2.The protein of claim 1, wherein the host cell is a mammalian cell. 3.The protein of claim 1, wherein the host cell has had a second DNAvector introduced into it, wherein said second expression vectorcomprises the following operably linked elements: a transcriptionpromoter; a DNA segment encoding an endoprotease; and a transcriptionterminator, wherein said host cell expresses the polypeptide encoded bythe DNA segment consisting of a nucleotide sequence shown in SEQ ID NO:1 from nucleotide 76 to nuclcotide 417; and said DNA segment encodingsaid endoprotease.